GTP-binding proteins represent a class of small, approximately 20 to 30 kDa, monomeric, receptor-coupled GTPases, which mediate signal transduction in eukaryotic cells. GTP-binding proteins act as molecular switches, alternating between an active GTP-bound state and an inactive GDP-bound state. Subfamily members of the GTP-binding proteins include Ras proteins, associated with the regulation of cell proliferation and differentiation, Rho and Rac proteins, associated with the regulation of cytoskeletal assembly, and Rab, Arf, Sar and Ran proteins, associated with the regulation of vesicular transport (Bourne et al. (1991), Nature. 349:117-127; Hall and Zerial (1995) General introduction. In: xe2x80x9cGuidebook to the Small GTPasesxe2x80x9d (M. Zerial and L. A. Huber Eds.), pp. 3-11, Sambrook and Tooze, Oxford University Press).
The Ras subfamily members share four conserved domains which, as demonstrated through both mutagenic studies and X-ray crystallography, are involved in the binding and hydrolysis of guanine nucleotides. Many Ras proteins also share a fifth conserved carboxy-terminal domain required for posttranslational modification of the Ras proteins prior to membrane localization (Boguski and McCormick (1993) Nature 366:643-654; Hall and Zerial (1995), supra).
At least nine of the sixty or so members of the Ras-related have greater than about 50% amino acid identity to H-, K-, and N-ras oncogenes (Hall and Zerial (1995), supra). These include R-ras1 and its related proteins R-ras-2(TC21) and the recently identified R-ras3 (Hall and Zerial (1995), supra; Kimmelmann et al. (1997), Oncogene 15(22):2675-2685) which share about 55% amino acid identity with H-ras, including an identical effector domain, and are about 70% identical to one another. The Rap proteins (Rap1a and b, Rap2a and b) also share a conserved effector domain and about 50% protein identity with H-ras. The RaIA and RaIB proteins also belong to the ras subfamily, with about 50% peptide identity to H-ras, although there is a one residue difference in their effector domain (Hall and Zerial (1995).
Recently two small GTPases, termed Rin and Rit, were identified. Rin and Rit share about 50% identity and four conserved GTP-binding motifs of Ras proteins,(Lee et al. (1996) J. Neurosci. 16(21):6784-6794). However, these two proteins were unusual in that they lacked the known CAAX recognition signal for C-terminal lipidation found in each of the other Ras subfamily members.
The Ras GTP-binding protein is coupled to a tyrosine kinase receptor. The formation of an agonist-receptor complex facilitates GTP binding to the Ras protein, whereupon the protein bound GTP is hydrolyzed to GDP via the intrinsic GTPase activity of the Ras protein. The GDP dissociates from the Ras protein and it reverts to its inactive form. This cycling between inactive and active states initiates a mitogen-activated kinase cascade which leads to the phosphorylation of a number of transcription factors in the nucleus which culminates in cell proliferation and differentiation.
GTP binding and its hydrolysis to GDP are catalyzed by at least two classes of proteins: guanine nucleotide exchange factors, which promote exchange between bound GDP and cytoplasmic GTP, and GTPase activating proteins, which stimulate the low intrinsic GTP hydrolysis by the GTPases (Boguski and McCormick (1993), Nature. 366, pp 643-654; Feig (1993), Science. 260, pp 767-768). Mutations in the GTP-binding proteins affecting the nucleotide exchange or GTP hydrolysis can stabilize the GTP- or GDP-bound conformation and thereby cause the GTP-binding proteins to be constitutively active or inactive. For example, Ras genes with point mutations are known, wherein the intrinsic GTPase activity of Ras GTP-binding protein is insensitive to GTPase activating proteins.
The link between mutations in Ras genes and cancer is well established. For example, there is a strong association between abnormal signal transduction involving activated Ras genes and the development of a variety of tumors. In fact, mutations in Ras genes are found in 30% of all human tumors and in some the mutation frequency approaches 100%, e.g., pancreatic adenocarcinoma. The number of genes encoding Ras or Ras-related proteins is unknown, but it is clear that their role in cellular processes is of crucial importance. Accordingly, the discovery of novel Ras-related genes will advance the understanding and treatment of human disease.
Relevant Literature
EST sequences present within the RAQ polynucleotide sequence of the invention are summarized in Table 1 below. The nucleotide residues of the RHOH sequence to which there is greater than 90% identity to a provided EST are indicated in the last column.
The sequence of human genes related to RAQ may be accessed at Genbank at the indicated accession numbers: R-ras2 (TC21), Genbank:M31468 Genbank:Y07565; Rin (human, Genbank accession #=Y07565); Rit, Genbank:U71203; RaIA, Genbank:X15014; and Rap1b, Genbank:X08004. The sequence of Dictostylium discoideum genes sharing homology with human RAQ may be accessed at Genbank at the indicated accession numbers: RasS, Genbank:Z14134; and RasD, Genbank:J04160.
The role of ras oncogenes in human cancer is reviewed in Zachos et al. (1997) Crit Rev Oncol Hematol 26(2):65-75 and in Bos (1989) Cancer Res. 49:4682-4689. The targeting of small GTPases for cancer therapies is discussed in Symons (1995) Curr. Opin. Biotechnol. 6:668-74.
The association of the 12p12-13 cytoband region with cancer is described in several references. The possibility of a human pulmonary adenoma susceptibility 1 (Pas1) locus homolog at 12p12-13 is discussed in Manenti et al. (1997) Carcinogenesis 18(10):1917-1920. Pas1 is a major locus affecting inherited predisposition to lung cancer in mice within the syntenic chromosome region. Johansson et al. ((1993) Genes, Chromosomes, Cancer. 8:205-218) and Hatta et al. ((1997) Br J Cancer. 75(9):1256-1262, suggested that a new tumor suppressor gene may lie within the 12p12-13 region.
The invention features polynucleotides encoding novel GTP-binding polypeptides, hereinafter referred to as RAQ polypeptides; expression vectors comprising a RAQ polynucleotide of the invention; isolated cells comprising a RAQ-encoding vector; a transgenic non-human animal comprising an alteration in a RAQ gene; and the use of RAQ polynucleotides in detecting in an individual the presence of a genetic polymorphism of a RAQ gene.
The invention further features novel RAQ polypeptides; monoclonal antibodies specific for RAQ polypeptides; and a method for making RAQ polypeptides.